Proteomic analysis of the Mexican lime tree response to "Candidatus Phytoplasma aurantifolia" infection

"Candidatus Phytoplasma aurantifolia" is the causative agent of witches' broom disease in the Mexican lime tree (Citrus aurantifolia L.), and is responsible for major tree losses in Southern Iran and Oman. The pathogen is strictly biotrophic, and, therefore, completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. We applied a proteomics approach to analyse gene expression in Mexican limes infected with "Ca. Phytoplasma aurantifolia". Leaf samples were collected from healthy and infected plants and were analysed using 2-DE coupled with MS. Among 800 leaf proteins that were detected reproducibly in eight biological replicates of healthy and eight biological replicates of infected plants, 55 showed a significant response to the disease. MS resulted in identification of 39 regulated proteins, which included proteins that were involved in oxidative stress defence, photosynthesis, metabolism, and the stress response. Our results provide the first proteomic view of the molecular basis of the infection process and identify genes that could help inhibit the effects of the pathogen.     

‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma aurantifolia’, New Phytoplasma Species Infecting Apple Trees in Iran

During several surveys in extensive areas in central Iran, apple trees showing phytoplasma diseases symptoms were observed. PCR tests using phytoplasma universal primer pairs P1A/P7A followed by R16F2n/R16R2 confirmed the association of phytoplasmas with symptomatic apple trees. Nested PCR using 16SrX group‐specific primer pair R16(X)F1/R1 and aster yellows group‐specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy indicated that apple phytoplasmas in these regions did not belong to the apple proliferation group, whereas aster yellows group‐related phytoplasmas caused disease on some trees. Restriction fragment length polymorphism (RFLP) analyses using four restriction enzymes (HhaI, HpaII, HaeIII and RsaI) and sequence analyses of partial 16S rRNA and rp genes demonstrated that apple phytoplasma isolates in the centre of Iran are related to ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma aurantifolia’. This is the first report of apples infected with ‘Ca. Phytoplasma asteris’ in Iran and the first record from association of ‘Ca. Phytoplasma aurantifolia’ with apples worldwide.     

Evaluation of anti-phytoplasma properties of surfactin and tetracycline towards lime witches' broom disease using real-time PCR

The anti-phytoplasma activities of surfactin (derived from Iranian native Bacillus subtilis isolates) and tetracycline towards Candidatus "Phytoplasma aurantifolia", the agent of lime Witches' broom disease, were investigated. HPLC was used to quantify the surfactin production in four previously characterized native surfactin-producing strains, and the one producing the highest amount of surfactin (about 1,500 mg/l) was selected and cultivated following optimized production and extraction protocols. Different combinations of purified surfactin and commercial tetracycline were injected into artificially phytoplasmainfected Mexican lime seedlings using a syringe injection system. An absolute quantitative real-time PCR system was developed to monitor the phytoplasma population shifts in the lime phloem during 3 months following the injections. The results revealed that the injections of surfactin or tetracycline had a significant inhibitory effect on Candidatus "P. aurantifolia". However, the combined treatment with both surfactin and tetracycline (1:1) resulted in the highest inhibition due to a synergic effect, which suppressed the phytoplasma population from about 2×10(5) to less than 10 phytoplasma units/g plant tissue.     

Arbuscular mycorrhizal colonization of lime in different agroecosystems of the dry tropics

 Tecomán, in the Mexican state of Colima, had the world's greatest production of lime Citrus aurantifolia Swingle. Typical farming systems in the area include: (a) high-input monoculture, (b) a high-input system in which lime trees grow together with coconut palms, (c) a low-input system called "Family Farms" or "Family Gardens". In the Family Gardens, cultural practices are minimal and other fruit trees (about 16 species) coexist with the lime trees. This traditional minimal input system makes use of locally available resources and they are structurally very diverse. Arbuscular mycorrhizae may be crucial for sustainable production in Family Gardens. Root colonization and spore populations of fungi were scored at 2-week intervals in the three agroecosystems during a 6-month period. First samples were taken after the application of chemical fertilizer and irrigation in the high-input systems. Root colonization of lime was much higher and consistent in the low-input plots than in conventionally farmed plots, with colonization levels of 50–62% that remained the same throughout the sampling time; the high-input systems showed a high variation and lower level of colonization, 36% and 27% in associated and monoculture systems, respectively. Spore abundance was higher in the high-input systems but showed constant variation. The results suggest a strong effect of agroecosystem on mycorrhizal colonization of lime roots. Accepted: 12 September 1995     

Molecular Characterization of Phytoplasmas Associated with Peach Diseases in Iran

Recently, peach trees showing leaf rolling, little leaf, rosetting, yellowing, bronzing of foliage and tattered and shot‐holed leaves symptoms were observed in peach growing areas in the central and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR using phytoplasma universal primer pairs P1/Tint, R16F2/R2, PA2F/R and NPA2F/R were employed to detect phytoplasmas. The nested PCR assays detected phytoplasma infections in 51% of symptomatic peach trees in the major peach production areas in East Azerbaijan, Isfahan, ChaharMahal‐O‐Bakhtiari and Tehran provinces. Restriction fragment length polymorphism (RFLP) analyses of 485 bp fragments amplified using primer pair NPA2F/R in nested PCR revealed that the phytoplasmas associated with infected peaches were genetically different and they were distinct from phytoplasmas that have been associated with peach and almond witches’‐broom diseases in the south of Iran. Sequence analyses of partial 16S rDNA and 16S–23S rDNA intergenic spacer regions demonstrated that ‘Candidatus Phytoplasma aurantifolia’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ are prevalent in peach growing areas in the central and north‐western regions of Iran.     

Association of two groups of phytoplasma with various symptoms in some wooden and herbaceous plants

During 2015–2016, wooden and herbaceous plants growing in parks, boulevards, fields, gardens and forests in Khuzestan province, southwestern Iran, were visually inspected for symptoms resembling phytoplasma. Fifty‐one symptomatic samples from nine different species and one symptomless sample from each plant were collected. Leaf midribs, petioles and the parts of stem cambium were separated and freeze‐dried. Total DNA was extracted using CTAB‐based method and tested for phytoplasma using a nested PCR assay. The expected size amplicons of 16S rDNA were sequenced and compared to those of reference phytoplasmas by BLASTn search and phylogenetic analysis. The consensus 16S rDNA sequence of the detected phytoplasma in narrow cattail related to reference phytoplasma group 16SrVI, “Candidatus Phytoplasma trifolii” while in the other plants were related to reference phytoplasma subgroup 16SrII–D, “Candidatus Phytoplasma aurantifolia.” All isolates showed 98%–99% sequence identity to members of their reference groups. To our knowledge, this is the first report of “Candidatus Phytoplasma aurantifolia”‐related strains infecting the plants of Acacia salicina, Alternanthera ficoidea, Melaleuca citrine, Citrus aurantium throughout the world and Celosia christata in Iran. Furthermore, this study is the first to report the association of a “Candidatus Phytoplasma trifolii”‐related strain with Typha angustifolia worldwide.     

Enhanced inhibition of Aspergillus niger on sedge (Lepironia articulata) treated with heat‐cured lime oil

Aims

This study aimed to examine heat curing effect (30–100°C) on antifungal activities of lime oil and its components (limonene, p‐cymene, β‐pinene and α‐pinene) at concentrations ranging from 100 to 300 μl ml^?1 against Aspergillus niger in microbiological medium and to optimize heat curing of lime oil for efficient mould control on sedge (Lepironia articulata).

Methods and Results

Broth dilution method was employed to determine lime oil minimum inhibitory concentration, which was at 90 μl ml^?1 with heat curing at 70°C. Limonene, a main component of lime oil, was an agent responsible for temperature dependencies of lime oil activities observed. Response surface methodology was used to construct the mathematical model describing a time period of zero mould growth on sedge as functions of heat curing temperature and lime oil concentration. Heat curing of 90 μl ml^?1 lime oil at 70°C extended a period of zero mould growth on sedge to 18 weeks under moist conditions.

Conclusions

Heat curing at 70°C best enhanced antifungal activity of lime oil against A. niger both in medium and on sedge.

Significance and Impact of the Study

Heat curing of lime oil has potential to be used to enhance the antifungal safety of sedge products.     

Molecular Characterization of Phytoplasmas Related to Apple Proliferation and Aster Yellows Groups Associated with Pear Decline Disease in Iran

Pear trees showing pear decline disease symptoms were observed in pear orchards in the centre and north of Iran. Detection of phytoplasmas using universal primer pair P1A/P7A followed by primer pair R16F2n/R16R2 in nested PCR confirmed association of phytoplasmas with diseased pear trees. However, PCR using group‐specific primer pairs R16(X)F1/R16(X)R1 and rp(I)F1A/rp(I)R1A showed that Iranian pear phytoplasmas are related to apple proliferation and aster yellows groups. Moreover, PCR results using primer pair ESFYf/ESFYr specific to 16SrX‐B subgroup indicated that ‘Ca. Phytoplasma prunorum’ is associated with pear decline disease in the north of Iran. RFLP analyses using HaeIII, HhaI, HinfI, HpaII and RsaI restriction enzymes confirmed the PCR results. Partial 16S rRNA, imp, rp and secY genes sequence analyses approved that ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma asteris’ cause pear decline disease in the centre of Iran, whereas ‘Ca. Phytoplasma prunorum’ causes disease in the north of Iran. This is the first report of the association of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma prunorum’ with pear decline disease worldwide.     

Identification of four distinct ‘Candidatus Phytoplasma’ species in pomegranate trees showing witches' broom,little leaf and yellowing in Jordan,and preliminary insights on their putative insect vectors and reservoir plants

During field surveys conducted in northern Jordan from June to November 2020, phytoplasma-like symptoms, including leaf yellowing/reddening and rolling, little leaf and witches' broom were observed in pomegranate. Disease incidence in 22 surveyed orchards ranged from 30% to 65%. Nested PCR-based amplification of 16S rRNA gene detected phytoplasmas in 17% of collected symptomatic pomegranate trees. Amplicon nucleotide sequence analyses allowed attributing the detected phytoplasmas to ‘Candidatus Phytoplasma solani’, ‘Ca. P. aurantifolia’, ‘Ca. P. asteris’ and ‘Ca. P. ulmi’. These phytoplasmas were found in plants showing specific symptoms and differentially distributed in the considered locations. Additionally, three cicadellids (Macrosteles sexnotatus, Cicadulina bipunctata and Psammotettix striatus) and two non-crop plants (Plantago major and Capsicum annuum) resulted hosting ‘Ca. P. asteris’ strains, and one cicadellid (Balclutha incisa) was carrying a ‘Ca. P. solani’ strain. A new pomegranate disease complex associated with multiple phytoplasmas, including ‘Ca. P. aurantifolia’ and ‘Ca. P. ulmi’, never reported before in this host plant, is described here. Moreover, preliminary indications are provided on its possible epidemiology in Jordan, involving two putative insect vectors (M. sexnotatus, B. incisa) first reported in the Country.     

Stable expression and phenotypic impact of <Emphasis Type="Italic">attacin E</Emphasis>transgene in orchard grown apple trees over a 12 year period

Background  

Transgenic trees currently are being produced by Agrobacterium -mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health risk of the modified plants, transgene stability over a prolonged period of time and the effect of the gene on tree and fruit characteristics. We studied the stability of expression and the effect on resistance to the fire blight disease of the lytic protein gene, attacin E, in the apple cultivar 'Galaxy' grown in the field for 12 years.     

Toward immunomodulation of witches broom disease of lime (WBDL) by targeting immunodominant membrane protein (IMP) of candidatus Phytoplasma aurantifolia

The witches' broom disease of lime (WBDL) caused by Candidatus Phytoplasma aurantifolia is the most devastating disease of acidian lime in southern part of Iran as it destroy thousands of trees yearly throughout these regions. Traditional methods such as eradication of infected trees and insect vector control have shown limited effect on this case. Therefore, alternative approaches such as plantibody-mediated resistance, have been considered. Throughout present study we prepared sufficient amount of antigen that is required for generation of specific monoclonal recombinant antibodies against Immunodominant membrane protein (IMP) which will be exploited for plantibody-mediated resistance approach. The gene encoding IMP protein was obtained by PCR amplification using specific primers and DNA extracted from the infected plants. Amplified fragment was then inserted into T/A cloning vector. Intact clones containing the right sequence was selected after digestion, PCR amplification and subsequent sequencing analysis. IMP encoding region having the right sequence was sub-cloned into pET28a bacterial expression vector. Large scale expression of His tagged recombinant protein was performed in the BL21-de3 strain of E. coli and purification under native conditions was carried out through immobilized metal ion affinity chromatography (IMAC) in a column containing Ni-NTA agarose beads. Successful expression and purification steps were confirmed by SDS-PAGE and western blotting analyses. The results obtained indicated the successful production of about 18 mg purified recombinant IMP protein with a low level of contamination in one liter cultured medium. Finally the purified protein was dialyzed in phosphate saline buffer and applied for immunization of mice.     

Transcriptional analysis of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> roots challenged with the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria bicolor</Emphasis>

Background  

Symbiotic ectomycorrhizal associations of fungi with forest trees play important and economically significant roles in the nutrition, growth and health of boreal forest trees, as well as in nutrient cycling. The ecology and physiology of ectomycorrhizal associations with Pinus sp are very well documented but very little is known about the molecular mechanisms behind these mutualistic interactions with gymnosperms as compared to angiosperms.     

Cytoplasmic- and extracellular-proteome analysis of <Emphasis Type="Italic">Diplodia seriata</Emphasis>: a phytopathogenic fungus involved in grapevine decline

Background  

The phytopathogenic fungus Diplodia seriata, whose genome remains unsequenced, produces severe infections in fruit trees (fruit blight) and grapevines. In this crop is recognized as one of the most prominent pathogens involved in grapevine trunk disease (or grapevine decline). This pathology can result in the death of adult plants and therefore it produces severe economical losses all around the world. To date no genes or proteins have been characterized in D. seriata that are involved in the pathogenicity process. In an effort to help identify potential gene products associated with pathogenicity and to gain a better understanding of the biology of D. seriata, we initiated a proteome-level study of the fungal mycelia and secretome.     

Identification of phytoplasmas associated with sesame phyllody disease in southeastern Iran

Phyllody disease is a threat to sesame production in Kerman province, southeastern Iran. RFLP analysis of PCR products of phytoplasma-specific 16S rRNA gene (1.8 kb) and phylogenetic analyses of 16S-23S rDNA spacer region (SR) sequence indicated that the predominant agent associated with sesame phyllody in Kerman province is a phytoplasma with 100% similarity with eggplant big bud, and peanut witches’-broom phytoplasmas, members of “Candidatus Phytoplasma aurantifolia” from Iran and China, respectively. Among the samples tested, only one strain (SPhSr1), had a unique RFLP profile and its SR was 100% similar in nucleotide sequence with the phytoplasma carried by Orosius albicinctus and Helianthus annus witches’-broom phytoplasma from Iran, members of “Ca. Phytoplasma trifolii”. Virtual RFLP patterns of SPhJ2 (representative of the predominant PCR-RFLP profiles) SR sequence were identical to those of peanut witches’-broom phytoplasma (16SrII-A, JX871467). However, SPhSr1 SR sequence patterns resemble (99.7%) those of vinca virescence phytoplasma (16SrVI-A, AY500817).     

Transmission of the Phytoplasma Associated with Bunchy Top Symptom of Papaya by Empoasca papayae Oman

Transmission tests were conducted with field‐collected Bunchy Top Symptoms (BTS) phytoplasma‐infected specimens of Empoasca papayae. BTS developed in all eight inoculated papayas 3 months later. The BTS phytoplasma was identified in six of eight inoculated papayas, whose partial 16S rRNA sequence (GenBank Accession no. FJ6492000 ) was 99.9% identical with those from the collected papayas (GenBank Accession no FJ649198 ) and E. papayae (GenBank Accession no. FJ649199 ), all of which are members of group 16SrII, ‘Candidatus Phytoplasma aurantifolia’. Results confirmed the ability of E. papayae to transmit the BTS phytoplasma.     

Survival,fecundity, and body mass of Amplicephalus curtulus influenced by ‘Candidatus Phytoplasma ulmi’ (16SrV‐A) infection

The leafhopper Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae) can transmit ‘Candidatus Phytoplasma ulmi’ (16SrV‐A) from a native Chilean shrub, Ugni molinae Turcz. (Myrtaceae), to ryegrasses. A recent study showed that this phytoplasma reduced the total protein content and the activity of detoxifying enzymes in A. curtulus, which could also affect its vector fitness. This study evaluated the effect of ‘Ca. Phytoplasma ulmi’ on the longevity, fecundity, and body mass of A. curtulus. Both females and males were exposed to ‘Ca. Phytoplasma ulmi’‐infected plants for 96 h, whereas a control group remained unexposed. Quartiles from adult emergence to 75% (t₇₅), 50% (t₅₀), and 25% (t₂₅) survival rates were determined for each leafhopper survival distribution. The dry weight was also established at the end of the experiment. The adult lifespan of phytoplasma‐infected males and females was significantly lower than that of the uninfected leafhoppers in quartile survival distributions t₅₀ and t₂₅. The phytoplasma‐infected males and females lived 3 and 4 weeks less than uninfected ones in the last quartile, respectively. Fecundity was established by number of nymphs per female (in four periods) in phytoplasma‐infected and uninfected assays. In general, the weekly pattern of the number of nymphs per phytoplasma‐infected female was lower than that of uninfected leafhoppers; it was 37% lower at the end of the experiment. Phytoplasma‐infected females weighed significantly less (11%) than uninfected individuals. Phytoplasma‐infected males weighed 8% less than uninfected ones, but this difference was not significant. Our data indicated that ‘Ca. Phytoplasma ulmi’ negatively affected the fitness of A. curtulus, and nymphs produced by phytoplasma‐infected females varied over time, which may influence the disease dynamics in nature or in field crops.     

Molecular identification of diverse ‘Candidatus Phytoplasma’ species associated with grapevine decline in Iran

Grapevine (Vitis vinifera) is one of the most important fruits in Iran where the provinces of Qazvin, Lorestan and Markazi are main producers. During 2013–2015, vineyards located in these provinces were surveyed to verify the presence of phytoplasma. The sample collection was based on symptomatology including decline, leaf yellowing and shortening of internodes. Total DNA was extracted from symptomatic and symptomless grapevine samples and used in nested‐polymerase chain reaction (PCR) assays with phytoplasma ribosomal primers (P1/Tint followed by R16F2n/R2, R16mF1/mR1, R16(I)F1/R1 or 6R758f/16R1232r). Nested‐PCR products were obtained only for symptomatic samples while samples from symptomless plants yielded no PCR products. Restriction fragment length polymorphism (RFLP) analyses with Tru1I, TaqI and Tsp509I and direct sequencing of amplicons followed by phylogenetic analyses indicated the presence of ‘Candidatus Phytoplasma fraxini’, ‘Ca. P. aurantifolia’, ‘Ca. P. solani’ and ‘Ca. P. phoenicium’‐related strains. In Marzaki province, there ‘Ca. P. aurantifolia’ strains were mainly detected, while in the other two provinces, all the four ‘Candidatus species’ were identified with the prevalence of ‘Ca. P. solani’‐related strains. In both provinces in one case, mixed phytoplasma infection was also detected by RFLP analyses. The presence of different phytoplasmas in positive samples indicates great phytosanitary significance due to grapevine economic importance for country. Grapevine phytoplasma infection represents a threat for other crops suggesting grapevine as alternative host species for the phytoplasmas already reported in Iran, while the ‘Ca. P. fraxini’ is for the first time identified in Iran.     

Detection and Molecular Identification of Aster Yellows Phytoplasma in Date Palm in Egypt

Phytoplasma‐like symptoms were detected in date palm trees (Phoenix dactylifera L.) in Al‐Giza Governorate in Egypt. Symptoms varied from leaf chlorotic streaks, stunting and marked reduction in fruit and stalk sizes. Direct and nested PCR of symptomatic samples using P1/P7 and R16F2n/R16R2n primers, respectively, of the 16S rRNA gene, resulted in a DNA amplification product of c. 1.3 kbp. Symptomless samples collected from the same location and the healthy control produced no product upon amplification. Products were cloned into TOPO TA vector for sequencing. Data generated were deposited in the GenBank (Accession KF826615 ). A BLAST search showed that the sequence of the 16SrRNA gene shared ‘Candidatus Phytoplasma asteris’ (16SrI group) with other isolates. Phylogenetic analysis revealed that the isolate clustered with the date palm phytoplasma causing Al‐Wijam disease in Saudi Arabia.